Enzymatic hydrolysis of cassava stems for butanol production of isolated Clostridium sp.

Saekhow, B.; Chookamlang, S.; Na-u-dom, A.; Leksawasdi, N.; Sanguanchaipaiwong, V. Article Enzymatic hydrolysis of cassava stems for butanol production of isolated Clostridium sp. Energy Reports Provided in Cooperation with: Elsevier Suggested Citation: Saekhow, B.; Chookamlang, S.; Na-u-dom, A.; Leksawasdi, N.; Sanguanchaipaiwong, V. (2020) : Enzymatic hydrolysis of cassava stems for butanol production of isolated Clostridium sp., Energy Reports, ISSN 2352-4847, Elsevier, Amsterdam, Vol. 6, Iss. 1, pp. 196-201, https://doi.org/10.1016/j.egyr.2019.08.042 This Version is available at: https://hdl.handle.net/10419/243732 Standard-Nutzungsbedingungen: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. 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If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ Available online at www.sciencedirect.com ScienceDirect Energy Reports 6 (2020) 196–201 www.elsevier.com/locate/egyr 6th International Conference on Energy and Environment Research, ICEER 2019, 22–25 July, University of Aveiro, Portugal Enzymatic hydrolysis of cassava stems for butanol production of isolated Clostridium sp. B. Saekhowa, S. Chookamlanga, A. Na-u-doma, N. Leksawasdib, V. Sanguanchaipaiwonga,∗ aDepartment of Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand bBioprocess Research Cluster, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University (CMU), Chiang Mai, 50100, Thailand Received 5 August 2019; accepted 22 August 2019 Abstract This research focused on the hydrolysis of cassava stems (CS) and subsequent utilization as a carbon source for the cultivation of isolated Clostridium sp. To yield the highest amount of reducing sugars (RS), the studies on the pretreatment with sodium hydroxide (NaOH) and the hydrolysis with cellulases, amylases, and mixed enzymes were carried out. Afterwards, the hydrolysate was utilized for the cultivation of isolated Clostridium sp. Experimental results revealed that CS after 1.0 M NaOH pretreatment at 121 ◦C for 15 min and cellulase hydrolysis (Accellerase®1500, 2500 CMC U/g CS) obtained 10.94 ±0.29 g/L RS concentration. Hydrolysis of CS with amylases (Termamyl®120, 1.2 U/g CS and AMG 300LTM 3.5 U/g CS) provided 34.85 ±0.75 g/L RS and the maximum RS amount of 47.90 ±0.39 g/L was obtained from the hydrolysis with mixed enzymes (Termamyl®120, 1.2 U/g CS, AMG 300LTM 3.5 U/g CS followed by Accellerase®1500, 2500 CMC U/g CS). From the cultivation of Clostridium sp. G10 using CS hydrolysate, the highest dry cell weight concentration of 1.28 ±0.07 g/L was obtained with 11.68 ±0.31 g/L butanol. It could be concluded that CS hydrolysate was comparable with glucose for utilization as a carbon source for butanol production. c 2019PublishedbyElsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the scientific committee of the 6th International Conference on Energy and Environment Research, ICEER 2019. Keywords: Acetone–butanol–ethanol fermentation; Anaerobe; Clostridium sp.; Cassava stem 1. Introduction Recently, lignocellulose materials from agricultural waste have gained increasing interest since they are plentiful and full of potential to utilize as a carbon source for microorganisms [1]. Moreover, the conventional disposal including burning and landfill have caused environmental problems and global warming. Cassava (Manihot esculenta) has been planted largely in Africa and Asia. Thailand is one of the top-5 five countries in term of ∗Corresponding author. E-mail address: [email protected] (V. Sanguanchaipaiwong). https://doi.org/10.1016/j.egyr.2019.08.042 2352-4847/ c 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). Peer-review under responsibility of the scientific committee of the 6th International Conference on Energy and Environment Research, ICEER 2019. B. Saekhow, S. Chookamlang, A. Na-u-dom et al. / Energy Reports 6 (2020) 196–201 197 production and net production value of cassava since 1975 [2] and in 2017 cassava production in this country reached 30.5 million tons [3]. Due to fuel crisis and insufficient resource of fossil fuel, the demand for alternative energy has been intensifying. Renewable carbon source is one of the distinct benefits of bioenergy and boosted the opportunity for generation of ecological and economical bio-fuel. Since its resemblance properties to gasoline, butanol has potential to be an alternative fuel. These resemblances include low vapour pressure (0.43 MJ/kg), energy level (29.2 MJ/L) and non-corrosivity [4]. Butanol is a precursor for many chemicals and solvent using in various industries [4] and can be produced using Clostridium sp. through acetone–butanol–ethanol (ABE) fermentation [5–7]. This metabolic fermentation is largely renowned due to the availability of wide range substrates and has been utilized in industrial process and patented [8]. Several publications examined the utilization of cassava stem (CS) for ethanol and biogas production [9–12], however, none of these reports focused on acetone–butanol–ethanol production. This is the first report to directly employ CS hydrolysate for Clostridium sp. for acetone–butanol–ethanol fermentation. In this research, CS was hydrolysed and effectively utilized for the cultivation of Clostridium sp. isolated from soil sediment in pond [13]. To obtain the highest reducing sugar concentration, CS was pretreated with NaOH prior to enzymatic hydrolysis using commercial amylase and cellulase. 2. Materials and methods 2.1. Cassava stem and bacterial strain Cassava ‘Rayong 81’ stem was obtained from Po-Phan sub-district, Na Chueak district, Mahasarakham province, Thailand. Subsequently, the stems were peeled and cut into 1-cm pieces, sun dried, as well as milled with a hammer miller (Retsch, SK100) Afterwards, the CS powder was screened through 300 µm (Mesh no. 50) sieve. The microorganism was isolated from soil sediment in pond [13] and identified as Clostridium sp. G10 (GenBank no. MG674699). It was transferred and maintained in Reinforced Clostridial Medium (RCM) (DifcoTM) every month at 4 ◦C and kept for long term storage in glycerol stock at −70 ◦C. 2.2. Pretreatment and enzymatic hydrolysis of cassava stem CS powder was treated with 1.0 M NaOH at the ratio of 1:10 (w/v) and autoclaved at 121 ◦C, 15 psi for 15 min. The mixture was filtered through Whatman®no. 1 filter paper and the filter cake was subsequently collected and washed until pH 7.0. For cellulase hydrolysis, the commercial Accellerase®1500 (1 mL/ 1 g of pretreated CS or 2500 CMC U/g CS) was added while pH level and the mixture volume were adjusted to 5.0 and 25 mL, respectively. Afterwards, the incubation was carried on at 50 ◦C for 48 h and the sample was collected every 12 h period. For the hydrolysis with α-amylase and amyloglucosidase, 15 g CS was added into 100 mL distilled water and autoclaved at 121 ◦C, 15 psi for 15 min. Addition of 0.05%(v/v) α-amylase (Termamyl®120, Sigma-Aldrich) of 3.3 mL volume (1.2 U/g CS) was done and incubated at 90 ◦C for 2 h in water bath (Memmert, WNE 45). The addition of 0.015%(v/v) amyloglucosidase (AMG 300LTM, Sigma-Aldrich) of 13.3 mL (3.5 U/g CS) was then followed before the incubation at 60 ◦C for 4 h. For mixed enzyme hydrolysis, the similar procedure was followed with simultaneous addition of Termamyl®120, AMG 300LTM, Accellerase®1500 with 48 h incubation period. In all hydrolysis experiments, the determination of reducing sugar concentration based on 3,5-dinitrosalicylic acid (DNS) method was followed [14]. 2.3. Preparation of medium, inoculum and ABE fermentation T6 medium [15] was implemented for inoculum preparation and ABE production which composed of 6 g/L tryptone, 2 g/L yeast extract, 0.5 g/L KH2PO4, 0.3 g/L MgSO4•7H2O, 0.01 g/L FeSO4•7H2O, 3 g/L ammonium acetate, 0.5 g/L cysteine hydrochloride and 50 g/L glucose. Glucose was separately sterilized from the other substituents at 121 ◦C, 15 psi for 15 min. The CS hydrolysate was supplemented with glucose to achieve 50 g/L of reducing sugar concentration. Clostridium sp. G10 in glycerol stock was transferred in RCM and heat-shocked at 80 ◦C for 10 min Herman et al. [16]. It was anaerobically incubated at 37 ◦C for 48 h and later transferred to T6 medium with 50 g/L glucose and cultivated in similar condition. This inoculum was transferred into T6 medium at 10% (v/v) and the anaerobic fermentation was performed in 500-mL flasks at 37 ◦C for 120 h with triplicate. Data was statistically analysed by SPSS for Windows program. 198 B. Saekhow, S. Chookamlang, A. Na-u-dom et al. / Energy Reports 6 (2020) 196–201 2.4. Chemical analyses All experiments were carried out in triplicate. Data was statistically analysed by SPSS for Windows program. The sampling was performed periodically and the solid part was used for determination of dry cell weight concentration. Afterwards, the culture samples were centrifuged at 5000 rpm (4200 ×g) for 10 min and the supernatants were collected for quantification of reducing sugar concentration using 3,5-dinitrosalicylic acid (DNS) method [14]. The concentration of glucose, maltose, xylose, cellobiose and ABE were determined by High Performance Liquid Chromatography (HPLC). The HPLC column was Aminex®fermentation monitor (150 mm ×7.8 mm). Refractive index detector was used and operated at 37 ◦C and 65 ◦C, respectively, with 5 mM H2SO4 as a mobile phase at 0.5 mL/min. The injection size was 20 µL. 3. Results and discussion 3.1. Pretreatment and enzymatic hydrolysis Alkali pretreatment have been a recognized process for lignin dissolving from lignocellulosic materials to obtain fermentable sugars [17]. Nevertheless, the reducing sugar concentration of merely 10.94 ±0.29 g/L was obtained from NaOH-pretreatment followed by Accellerase®1500 hydrolysis of CS for 48 h (Fig. 1a). Considering that CS contained 15%–34% hemicellulose and 35%–42% cellulose depending on variety and plantation area [18], the result implied that abundant glucan was absent. There was current report regarding high starch substance up to 42% of dry CS weight [10]. To hydrolyse starch, NaOH pretreatment was neglected and amylase enzymes (Termamyl® 120 and AMG 300LTM) were added to examine the glucan hydrolysis process. Fig. 1b showed the reducing sugar concentration of 34.85 ±0.75 g/L from the addition of Termamyl®120 (2 h) and AMG 300LTM (4 h). The final procedure included the addition of Termamyl®120 (2 h) and AMG 300LTM with Accellerase®1500 (48 h) resulting in the reducing sugar concentration of 47.90 ±0.39 g/L at 12 h (Fig. 1c). This value has no significant difference (p>0.05) from the reducing sugar concentration of 47.18 ±0.73 g/L at 38 h. Thus, the addition of Termamyl®120 (2 h) and AMG 300LTM with Accellerase®1500 (12 h) was selected to prepare for ABE fermentation medium. Fig. 1. Time course of the reducing sugar concentration from cassava stem measured by DNS method with (a) NaOH pretreatment and cellulase hydrolysis at 50 ◦C for 48 h, (b) Termamyl®120 at 90 ◦C for 2 h and AMG 300LTM hydrolysis at 60 ◦C for 4 h, (c) Termamyl® 120 at 90 ◦C for 2 h and AMG 300LTM with cellulase hydrolysis at 50 ◦C for 48 h and (d) the concentration of glucose, maltose, xylose and cellobiose of cassava stem hydrolysate using Termamyl®120 and AMG 300LTM with Accellerase®1500 determined by HPLC. B. Saekhow, S. Chookamlang, A. Na-u-dom et al. / Energy Reports 6 (2020) 196–201 199 Fig. 2. Time course of dry cell weight concentration from Clostridium sp. G10 cultivation in T6 medium containing glucose (blue square) and cassava stem hydrolysate (red triangle) at 37 ◦C for 120 h under anaerobic condition. From HPLC analysis (Fig. 1d), the CS hydrolysate contained 29.94 ±0.37 g/L glucose, 3.54 ±0.06 g/L maltose and 2.30 ±0.05 g/L xylose. Small amount of xylose suggested that ‘Rayong 81’ stem contained small amount of hemicellulose. To compare the reducing sugar concentration, Table 1 indicated the concentration of sugars from CS with different pretreatment and enzymatic hydrolysis from various publications. Table 1. The concentration of sugars from cassava stem with different pretreatment and enzymatic hydrolysis. Conditions Cassava stem concentration (%w/v) The sugar concentration Reference Pretreatment Enzymatic hydrolysis Reducing sugars Glucose (g/L) Thermohydrolysis (210 ◦C, 45 min) – 10 14.91 (g/L) – Nanssou et al. [19] Microwave assisted NaOH (140 W, 30 min) amylase (200U/g) amyloglucosidase (70 U/g) cellulose (20 FPU/g) β-glucosidase (1 IU/g) 5 459.0 (mg/g CS) – Sudha et al. [20] Diluted H2SO4 (170 ◦C, 20 min) Liquozyme SC4X (0.01 mL /g) Spirizyme Excel XHS (0.015 mL/g) 10 – 29.9 Martin et al. [21] Microwave assisted NaOH (300 W, 7 min) Cellic®CTec2 (0.005 g enz. prot./ mL) Optimash BG (0.005 mL/ mL) StargenTM002 (0.001 mL/ mL) 10 59.05 (g/L) – Pooja et al. [11] Repeated H2SO4 hydrolysis (111 ◦C, 1 h & 1 h) 40 133.17 (g/L) [glucose + xylose] 94.05 Tanaka et al. [12] – Termamyl®120 (1.2 U/g) AMG 300LTM (3.5 U/g) Accellerase®1500 (2500 CMC U/g) 10 47.90 (g/L) 29.94 This report 3.2. Effect of cassava stem hydrolysate on cell growth and ABE production The time profiles of G10 strain growth has been shown in Fig. 2. The cultivation in T6 medium with 50 g/L glucose contributed the highest dry cell weight of 1.45 g/L at 38 h. The dry cell weight in CS hydrolysate with the equivalent reducing sugar concentration of 50 g/L was 1.28 g/L after 62 h of cultivation. There was no obvious difference of dry cell weight between the cultivation of Clostridium sp. G10 using CS hydrolysate and glucose. On the other hand, there was slightly difference when compared the solvent production of Clostridium sp. G10 utilizing different carbon source (Fig. 3). From the cultivation of Clostridium sp. G10 using CS using glucose provided the maximum butanol concentration of 12.15 ±0.07 g/L. No publication report has directly utilized CS hydrolysate as a carbon source for acetone–butanol–ethanol fermentation. Torres et al. [22] reported hydrogen production from sludge in anaerobic fixed-bed reactor. Since the mixed culture was utilized, 1.56 g/L butanol was also produced from cassava starch wastewater with total carbohydrate of 2.12 g/L. The butanol concentration of 3.365 g/L was obtained from the production medium containing 60 g/L acid-hydrolysed sago (made from cassava starch) waste residues using C. bifermentans PNAS1 [23]. 200 B. Saekhow, S. Chookamlang, A. Na-u-dom et al. / Energy Reports 6 (2020) 196–201 Fig. 3. Time course of the acetone (brown cross), butanol (purple diamond) and ethanol (green circle) concentration in T6 medium containing (a) glucose; (b) cassava stem hydrolysate from Clostridium sp. G10 at 37 ◦C with anaerobic condition. 4. Conclusion In this research, cassava stem, agricultural waste, was utilized as a part of carbon source for isolated Clostridium sp. G10. Prior to the bacterial cultivation, the cassava stem was effectively hydrolysed by mixed enzymes (Termamyl®120, AMG 300LTM, Accellerase®1500) and 47.90 g/L reducing sugar concentration was obtained. The concentrations of butanol achieved from T6 medium containing glucose (12.15 g/L) and cassava stem hydrolysate (11.68 g/L) were not significantly different (p>0.05). Renewable cassava stem has capability for the fermentation of cost-effective and sufficient biobutanol. This report has been the first to straightforwardly utilize cassava stem for butanol production. Acknowledgements The authors would like to acknowledge the financial support from Faculty of Science, KMITL (V. Sanguanchaipaiwong, grant numbers 2562-01-05-14) as well as National Research Council of Thailand (NRCT) and Chiang Mai University (N. Leksawasdi) for funding this research. References [1] Saini Jitendra Kumar, Saini Reetu, Tewari Lakshimi. 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